Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression

Abstract

During mitosis, the recruitment of spindle-checkpoint-associated proteins to the kinetochore occurs in a defined order. The protein kinase Bub1 localizes to the kinetochore very early during mitosis, followed by Cenp-F, BubR1, Cenp-E and finally Mad2. Using RNA interference, we have investigated whether this order of binding reflects a level of dependency in human somatic cells. Specifically, we show that Bub1 plays a key role in the assembly of checkpoint proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of Bub1. Repression of Bub1 increases the number of cells with lagging chromosomes at metaphase, suggesting that Bub1 plays a role in chromosome congression. However, repression of Bub1 does not appear to compromise spindle checkpoint function either during normal mitosis or in response to spindle damage. This raises the possibility that, in the absence of Bub1, other mechanisms contribute to spindle checkpoint function.

Bub1: Escapades in a Cellular World

“…22,29 A likely reconciliation of these results may emerge when considering the different degrees of Bub1 depletion by RNAi. Thus, a partial Bub1 depletion led to failed congression and activation of a spindle checkpoint arrest, 22 whereas a more complete depletion eliminated spindle checkpoint function. 29 bub1 LInKs TO cIn, p53, sV40 And cAnceR Much enthusiasm stems from the original demonstration that Bub1 mutations can be found in colorectal carcinomas exhibiting chromosomal instability (CIN), which is a state where whole chromosomes are gained or lost.…”

“…21 Studies with RNAi or antibody depletion in Xenopus extracts indicate that Bub1 plays an key role for recruitment of other checkpoint and motor proteins such as Mad1, Mad2, BubR1, CENP-E and Plk1. 16,22,23 Xenopus extract depletion experiments clearly demonstrate that Bub1 plays a fundamental role for assembly of a functional inner centromere, in fact acting as a "master organizer". 24 These studies directly show that Bub1, in a kinase-dependent manner, is important for proper localization of the chromosomal passenger complex that includes Aurora B, Survivin and INCENP.…”

“…23 In addition to the roles of Bub1 in the spindle checkpoint, centromere structure, and sister chromatid cohesion, it was also observed that Bub1 is required for normal metaphase congression. 22,29 RNAi studies initially led to conflicting views regarding the requirement of Bub1 in the spindle checkpoint. 22,29 A likely reconciliation of these results may emerge when considering the different degrees of Bub1 depletion by RNAi.…”

“…22,29 RNAi studies initially led to conflicting views regarding the requirement of Bub1 in the spindle checkpoint. 22,29 A likely reconciliation of these results may emerge when considering the different degrees of Bub1 depletion by RNAi. Thus, a partial Bub1 depletion led to failed congression and activation of a spindle checkpoint arrest, 22 whereas a more complete depletion eliminated spindle checkpoint function.…”

The Spindle Assembly Checkpoint: Clock or Domino?

“…Specifically, it phosphorylates the microtubule-binding KMN network at kinetochores in a proximity-dependent manner if kinetochores are not stretched apart through a bipolar attachment Welburn et al 2010). But it has also been implicated directly in the spindle checkpoint signaling, as it contributes to the loading of Mad2 to kinetochores in human cells and is essential for the spindle checkpoint in S. pombe (Johnson et al 2004;Vanoosthuyse and Hardwick 2009). Given that Ipl1/Ark1 is essential in fungi, it is not considered a checkpoint protein.…”